Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is still one of the most deadly diseases afflicting the world's population, therefore novel anti-TB therapeutics are urgently needed. Iron is an essential for Mtb survival, which it must sequester from its host. These proposed studies center around the newly discovered, novel mycobacterial heme-iron acquisition pathway. Recently, an Mtb cytosolic heme-degrading protein, MhuD, has been characterized, whereby MhuD catalyzes the release of iron from heme by cleavage of the heme tetrapyrrole ring. However, an Mtb mhuD deletion mutant did not completely abolish growth in the presence of heme, suggesting that another undiscovered protein is also functioning to release iron from heme. We propose that Mtb dye-decolorizing peroxidase, Mtb Dyp, may abstract iron from heme without the cleavage of the tetrapyrrole ring, as Mtb Dyp is a close homolog to E. coli proteins which function in this manner. Furthermore, Mtb also harbors a secreted encapsulin homolog (Enc), forming a two-gene operon with Mtb Dyp. These proteins are proposed to interact via an extended C-terminal extension in Mtb Dyp, whereby Mtb Dyp is encapsulated by Mtb Enc nano-cage. Interestingly, we have also observed that an Mtb iron-binding ferritin protein, Mtb BfrB, also contains an extended C-terminal tail, and may indicate that Mtb BfrB is also be compartmentalized by Mtb Enc. This proposal outlines a pragmatic structural, biochemical and genetic approach to characterize the heme-degrading function of Mtb Dyp, along with its interaction with Mtb Enc. Thus results from this study will not only further our understanding of Mtb heme-iron uptake and utilization but will also provide new candidates, Mtb Dyp and Mtb Enc, which have no eukaryotic homologues, for the development of innovative anti-TB therapeutics. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is still one of the most deadly diseases afflicting the world's population therefore there is an urgent need to develop novel anti-TB therapeutics. This proposal seeks to address this challenge through characterizing two proteins, Mtb Dyp and Mtb Enc, which have no eukaryotic homologues and are proposed to be involved in the novel mycobacterial heme uptake pathway. The results from this structural, functional, and genetic study of these proteins may set the stage for the discovery of an entirely new class of anti-TB therapeutics and potential vaccine candidates, which has the potential to have an extraordinary impact on global health.